There is growing awareness that androgens and estrogens have general metabolic roles that are not directly involved in reproductive processes. These include actions on vascular function, lipid and carbohydrate metabolism, as well as bone mineralization and epiphyseal closure in both sexes. In postmenopausal women, as in men, estrogen is no longer solely an endocrine factor but instead is produced in a number of extragonadal sites and acts locally at these sites in a paracrine and intracrine fashion. These sites include breast, bone, vasculature, and brain. Within these sites, aromatase action can generate high levels of estradiol locally without significantly affecting circulating levels. Circulating C19 steroid precursors are essential substrates for extragonadal estrogen synthesis. The levels of these androgenic precursors decline markedly with advancing age in women, possible from the mid-to-late reproductive years. This may be a fundamental reason why women are at increased risk for bone mineral loss and fracture, and possibly decline of cognitive function, compared with men. Aromatase expression in these various sites is under the control of tissue-specific promotors regulated by different cohorts of transcription factors. Thus in principle, it should be possible to develop selective aromatase modulators (SAMs) that block aromatase expression, for example, in breast, but allow unimpaired estrogen synthesis in other tissues such as bone.

It is well established that spermatogenesis is controlled by gonadotrophins and testosterone. However, a role for estrogens in male reproduction recently was suggested in adult mice deficient in estrogen receptor ␣. . Despite the demonstration of the aromatase enzyme, which converts androgens to estrogens, and estrogen receptors within the rodent seminiferous epithelium, the role of aromatase and estrogen in germ cell development is unknown. We have investigated spermatogenesis in mice that lack aromatase because of the targeted disruption of the cyp19 gene (ArKO). Male mice deficient in aromatase were initially fertile but developed progressive infertility, until their ability to sire pups was severely impaired. The mice deficient in aromatase developed disruptions to spermatogenesis between 4.5 months and 1 year, despite no decreases in gonadotrophins or androgens. Spermatogenesis primarily was arrested at early spermiogenic stages, as characterized by an increase in apoptosis and the appearance of multinucleated cells, and there was a significant reduction in round and elongated spermatids, but no changes in Sertoli cells and earlier germ cells. In addition, Leydig cell hyperplasia͞hypertrophy was evident, presumably as a consequence of increased circulating luteinizing hormone. Our findings indicate that local expression of aromatase is essential for spermatogenesis and provide evidence for a direct action of estrogen on male germ cell development and thus fertility.

The regulation of GnRH neurons by kisspeptin is critical for normal puberty onset in mammals. In the rodent the kisspeptin neurons innervating GnRH neurons are thought to reside in the rostral periventricular area of the third ventricle (RP3V). Using kisspeptin immunocytochemistry we show that kisspeptin peptide expression in the RP3V of female mice begins around postnatal d 15 (P15) and rapidly increases to achieve adult-like levels by P30, the time of puberty onset. Ovariectomy of female pups at P15 resulted in a 70-90% reduction (P < 0.01) in kisspeptin peptide expression within the RP3V of P30 or P60 mice. Replacement of 17-beta-estradiol (E2) in P15-ovariectomized mice from P15-30 or P22-30 resulted in a complete restoration of kisspeptin peptide expression in the RP3V (P < 0.01). Kisspeptin-immunoreactive fibers throughout the hypothalamus, including the arcuate nucleus, followed the same pattern of estrogen-dependent expression. To test the absolute necessity of estrogen for kisspeptin expression in the RP3V, aromatase knockout mice were examined. Kisspeptin-immunoreactive cells were detected in the arcuate nucleus, but there was a complete absence of kisspeptin peptide in RP3V neurons of aromatase knockout adult females. These results demonstrate that E2 is essential for the prepubertal development of kisspeptin peptide within RP3V neurons and suggest that an E2-kisspeptin positive feedback mechanism exists before puberty. This implies that RP3V kisspeptin neurons are E2-dependent amplifiers of GnRH neuron activity in the prepubertal period.

IntroductionIn the last decade or so, our knowledge of the roles of the steroids referred to as 'sex hormones' namely testosterone and estradiol, has undergone a quiet revolution. In the first place, models of androgen and estrogen insufficiency, whether they be natural or engineered, have revealed new and unexpected roles for sex steroids, some of which have nothing to do with reproduction.Furthermore both categories of steroid have roles in both sexes which blunts the definition of the terms 'androgen' and 'estrogen'. Secondly, the gradual acceptance of the role of local steroid hormone action, particularly as it applies in postmenopausal women and in men, provides new insights into the significance of paracrine and intracrine actions, and requires a re-evaluation of the importance of circulating steroid hormone levels. This article will attempt to review both of these developments, particularly in the context of the work of this laboratory on the aromatase knock-out (ArKO) mouse and the role of local aromatase expression within the breast and breast cancer. The concept of local estrogen biosynthesisModels of estrogen insufficiency have revealed new and unexpected roles for estrogens in both males and females. These models include natural mutations in the aromatase gene, as well as mouse knockouts of aromatase and the estrogen receptors (1)(2)(3)(4)(5)(6) . In addition there is one man described with a natural mutation in the ERα (7) . Some of the roles of estrogens apply equally to males and females and do not relate to reproduction, for example the bone, vascular and Metabolic Syndrome phenotypes.In postmenopausal women and in men, estradiol does not appear to function as a circulating hormone, instead it is synthesised in a number of extragonadal sites such as breast, brain and bone where its actions are mainly at the local 2 level as a paracrine or intracrine factor. Thus in postmenopausal women and in men, circulating estrogens are not the drivers of estrogen action, rather they reflect the metabolism of estrogens formed in these extragonadal sites, they are reactive rather than proactive (8) . Importantly, estrogen biosynthesis in these sites depends on a circulating source of androgenic precursors such as testosterone. Table 1 shows the plasma steroid levels in postmenopausal women and in men. As can be seen, the levels of estrone and estradiol in the plasma of postmenopausal women are extremely low, lower in fact than those in the circulation of men; and moreover the levels of circulating testosterone are an order of magnitude greater than those of estrogens in postmenopausal women.This in itself would suggest that circulating testosterone is better placed to serve as a precursor of estradiol in target tissues than is circulating estradiol.On the other hand, the levels of testosterone in the blood of men are an order of magnitude higher than those of women. Significantly, levels of DHEA and DHEAS in the blood of both men and women are orders of magnitude higher than those of the circulating active ste...

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Estrogen deficiency in the aromatase knockout (ArKO) mouse leads to the development of obesity by as early as 3 months of age, which is characterized by a marked increase in the weights of gonadal and infrarenal fat pads. Humans with natural mutations of the aromatase gene also develop a metabolic syndrome. In the present study cellular and molecular parameters were investigated in gonadal adipose tissue from 10-wk-old wild-type (WT) and ArKO female mice treated with 17beta-estradiol or placebo to identify the basis for the increase in intraabdominal obesity. Stereological examination revealed that adipocytes isolated from ArKO mice were significantly larger and more abundant than adipocytes isolated from WT mice. Upon treatment with estrogen, the volume of these adipocytes was greatly reduced, whereas the reduction in the number of adipocytes was much less pronounced. Transcriptional analysis using real-time PCR revealed concomitant changes with adipocyte volume in the levels of transcripts encoding leptin and lipoprotein lipase, whereas peroxisome proliferator-activated receptor gamma levels followed a pattern closer to that of adipocyte number. Little change was observed in levels of transcripts for factors involved in de novo fatty acid synthesis, beta-oxidation, and lipolysis, suggesting that changes in the uptake of lipids from the circulation are the main mechanisms by which estrogen regulates lipid metabolism in these mice.

Dysfunctional dopaminergic (DAergic) neurotransmission between the substantia nigra pars compacta (SNc) and the dorsal striatum (the nigrostriatal pathway) causes several prominent movement disorders (e.g. the motor symptoms of Parkinson's disease, dystonias and dyskinesias). Both too much and too little DA signalling can be problematic and nigrostriatal DAergic transmission is maintained at normal levels by several homeostatic mechanisms acting over timescales of milliseconds to weeks: e.g. binding of DA to tyrosine 3-monooxygenase (EC 1.14.16.2) (tyrosine Received November 1, 2010; revised manuscript received December 13, 2010; accepted December 14, 2010. Address correspondence and reprint requests to T. D. Aumann, Florey Neuroscience Institutes, The University of Melbourne, Parkville, Victoria, Australia 3010. E-mail: tim.aumann@florey.edu.auAbbreviations used: D2R, D 2 dopamine receptor; DA, dopamine; EGF, epidermal growth factor; FGF, fibroblast growth factor; GDNF, glial cellderived neurotrophic factor; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RRF, retrorubral field; SDS, sodium dodecyl sulfate; SK, small-conductance, Ca 2+ -activated potassium; SNc, substantia nigra pars compacta; TH, tyrosine hydroxylase; TTX, tetrodotoxin; VTA, ventral tegmental area. AbstractStriatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1 lM tetrodotoxin decreased TH expression beginning around 20 h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca 2+ -activated potassium channels with 300 nM apamin increased TH mRNA and protein between 12 and 24 h later in slices of adult midbrain. Twoweek infusions of 300 nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca 2+ channels, GABA A receptors, high K + , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change 20 h following sustained chang...